Inflammation at wound sites and sites of infection is often characterized by, inter alia, a strong infiltration of leukocytes at the site of inflammation. In particular, polymorphonuclear cells (PMN) are the predominant cell type recovered from the sites of inflammation, such as inflammatory joints (inflamed intraarticular and periarticular spaces) (Terkeltaub, 1992; Dieppe et al., 1979).
Inflammation can be reduced by the action of, for instance, such anti-inflammatory agents as glucocorticoids, produced by the body in response to inflammation. One of the many actions carried out by glucocorticoids is the induction of lipocortin 1 (LC1), which itself inhibits arachidonic acid release and cell proliferation (processes usually associated with inflammation).
The reviewed experimental evidence (Flower and Rothwell, 1994) supports the concept that lipocortin (LC) 1 is a key mediator of many effects of glucocorticoids including the suppression of lipid mediator release (Cirino et al., 1987) the inhibition of fever, (Carey et al., 1990; Davidson et al., 1991), paw oedema (Cirino et al., 1989) and polymorphonuclear leukocyte (PMN) migration (Perretti et al., 1993), the inhibition of the release of adrenocorticotrophic hormone (ACTH) (Taylor et al., 1993) and other anterior pituitary hormones (e.g. Taylor et al., 1993, 1995) and the inhibition of the induction by endotoxin of nitric oxide synthase (Wu et al., 1995).
LC1 is a member of a super-family of proteins termed the annexins (reviewed by Raynal and Pollard, 1994). Members of this protein group are identified by a common structural motif comprising four repeating subunits (in some members of the family, eight repeating subunits). While this core domain is highly conserved amongst members of the annexin family each of the individual proteins has a distinct N-terminal domain of variable length and it has been suggested that since this is a distinguishing feature, it probably contributes to the biological activity specifically associated with each member. Indeed, previous work has demonstrated that LC1 lacking the N-terminal domain is without activity in some assays of inflammation and mediator release, whereas the full length N-terminus N-acetyl LC12-26 is biologically active in several systems (Cirino et al., 1993; Perretti, 1994).
The A549 cell line is a useful model for studying LC1 biology. The inhibitory action of glucocorticoids on cell proliferation in this model seems to be mediated by the induction and externalization of LC1, which subsequently impairs arachidonic acid release and therefore the release of eicosanoids which function as autocrine growth stimulators in this cell system (Croxtall and Flower, 1992). The glucocorticoid block of arachidonic acid release and cell growth may be neutralized by anti-LC1 neutralizing monoclonal antibodies (Croxtall and Flower, 1992; Croxtall et al., 1995) or antisense deoxynucleotides (Croxtall and Flower, 1994), thus confirming the central role for this protein in glucocorticoid action.
In previous publications (Croxtall et al., 1993), it has been demonstrated that the N-terminal domain of LC1 is crucial in exerting this inhibitory effect on A549 cell function and that this biological property seems to reside in the downstream portion of the N-terminal domain (LC113-25) as LC1 1-12 is inactive in this model.
To define further the region necessary for the biological activity of the lipocortin N-terminal domain, experiments were carried out where a family of 25 peptides was synthesized in which systematic deletions were made from the N- and C-termini. This enabled a search to be carried out with more precision for the biological active region of the molecule (Croxtall et al., 1998). The results of these studies highlighted the importance of the domain EQEYV (SEQ ID NO:1), as a highly conserved sequence presenting all active peptides. The shortest peptide, which produced significant inhibitory activity, was LC118-25 (EQEYVQTV (SEQ ID NO:2)), implying that the domain EQEYV (SEQ ID NO:1), while essential, was not sufficient for biological activity.
However, the studies carried out by Croxtall and colleagues (Croxtall et al., 1998) were based upon an in vitro assay where cell division was measured.
Surprisingly, it has now been found that the in vivo anti-inflammatory properties of LC1 are contained within a different part of the N-terminal amino acid sequence of LC1, specifically LC12-6, (N-acetyl LC12-6=AMVSE (SEQ ID NO:3)).